ABSTRACT It is increasingly appreciated that preserving intestinal barrier function is essential for maintaining gut homeostasis. Disruption of this barrier drives chronic intestinal inflammation as observed in inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC). As part of this barrier the mucus gel layer (MGL) lining the intestinal epithelium plays a vital role in controlling tissue homeostasis, in part by exclusion of bacteria. We have identified epithelial A2B adenosine receptor signaling as a mechanism to restore intestinal barrier function. Investigations into the mechanism by which the epithelial A2B receptor functions to protect this barrier identified a potential novel role for A2B receptor regulation of the MGL. Epithelial A2B receptor signaling did not affect Muc2 expression, the predominant component of the MGL. Instead we identified that the secreted mucin, Muc5ac, was elevated in murine colitis and A2B deficient mice failed to upregulate Muc5ac despite having exacerbated inflammation and injury. A2B signaling increases MUC5AC mRNA expression in human intestinal epithelial cells and prevents bacterial translocation across the epithelium in vitro. MUC5AC expression is enhanced in the epithelium of both CD and UC patients. To date nothing is known about the role of MUC5AC in either CD or UC. Utilizing mice with genetic deletion of Muc5ac (Muc5ac-/-) we observed more severe colitis-associated injury in Muc5ac-/- mice compared to wildtype controls. This was associated with exacerbated anti-bacterial responses in the colon of Muc5ac-/- mice. 70% of CD patients present will ileal involvement. To investigate if the function of Muc5ac might have a role in CD or if the protective effect we observed is limited to the colon we utilized a Crohn's-like model of ileitis in which Muc5ac is deleted. These mice demonstrate increased bacterial translocation to the mesenteric lymph nodes and increased active ileal inflammation compared to ileitis controls. This data suggests that Muc5ac expression may limit host-bacteria interactions to protect the intestinal barrier during inflammation. We hypothesize that A2B epithelial signaling drives Muc5ac/MUC5AC expression in the intestinal epithelium to suppress epithelial:bacteria contact during inflammation as observed in IBD.